70 results about "Nicking enzyme" patented technology

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.

The invention relates to a manufacturing method and application of an electrochemiluminescence sensor for detecting thrombin. Ruthenium bipyridine with active group is modified to the gold nanoparticle surface to form an electrochemiluminescence nanoparticle probe, which plays the role of amplifying electrochemiluminescence, and the upper part DNA single strand is modified on the working electrode surface to constitute an electrochemiluminescence sensor. The target analyte of thrombin is subjected to reaction with partial DNA double strands and then with DNA polymerase, Nb.BbvCI nicking enzyme and partial DNA single strand to obtain DNA hybrid solution which is added into the sensor, so as to increase electrochemiluminescence nanoparticle probes on the electrode surface and further lead to an enhanced electrochemiluminescence signal, and accordingly thrombin determination is realized. The inventive sensor has high selectivity and detection sensitivity.

The invention discloses a cascading isothermal amplification based miRNA fluorescence detection kit and a miRNA fluorescence detection method. The target miRNA can specifically recognize and form a stable three-way crossing structure with a ternary probe through complementary hybridization of nucleic acids; a ternary primer in the three-way crossing structure performs strand displacement amplification (SDA) along a ternary template and generates a number of SDA template products; the SDA template product can open a difunctional hairpin probe to trigger nickase to perform signal amplification reaction finally; and a cyclophorase digestion molecular beacon can generate cascading amplified fluorescence signals. The kit can detect miRNA low to 100fM; and the detection method has superhigh specificity, can well distinguish highly homologous sequences at different mispairing positions and solves the difficult problem of false positive result caused by poor specificity in the existing detection method.

An engineered highly specific DNA-cleavage enzyme delivers a site-specific nick in a double stranded DNA, to cleave one DNA strand within its target site while leaving the opposing DNA strand intact. The engineered enzyme provides the ability to induce a gene conversion event in a mammalian cell. An engineered sequence-specific nickase derived from a LAGLIDADG homing endonuclease is altered by a single amino acid residue, wherein the amino acid residue is involved in the polarization of solvent molecules and acid-base catalysis in the active site without affecting direct contacts between the enzyme and either the bound DNA or bound metal ions. Engineered, site-specific nickase variants, such as of I-AniI and other homing endonucleases, are particularly useful in targeted genome engineering as well as therapeutic, targeted gene repair.

The present invention provides a simple method for producing a dumbbell-shaped DNA.

A method for producing a dumbbell-shaped DNA, wherein each of sense and antisense strands is connected at both the 5′ and 3′ ends of a linear-shaped double stranded DNA by a single stranded DNA of loop structure, comprising the steps of;

• 1) amplifying a target DNA in a template DNA by PCR using sense and antisense primers, wherein each of the sense and antisense primers contains the following sequence (a) at the 5′ end and also contains the following sequences (b), (c), and (d) in order from the 5′ end to the 3′ end,
  • (a) a part of a sense sequence of a nickase recognition sequence, comprising the sequence of a region between the site where a nick is introduced by the action of a nickase and the 3′ end,
  • (b) a sequence capable of forming a loop structure from a single strand,
  • (c) the entire antisense sequence of the nickase recognition sequence (a),
  • (d) a sequence complementary to all or part of the sequence of the target DNA;
• 2) treating the amplified DNA product of step 1) with a nickase of (a);
• 3) heating and then annealing the nickase treated amplified DNA product of step 2); and
• 4) treating the heated and annealed amplified DNA product of step 3) with DNA ligase, wherein the sense and antisense primers used in step 1) are phosphorylated at the 5′ end, or the amplified DNA product is phosphorylated at the 5′ end after step 1) but before step 4).

The invention relates to a novel method for isothermal amplification detection of a double-stranded nucleic acid based on nicking enzyme, belongs to the field of nucleic acid test, and in particular to a method for amplifying a single-chain target from a double-stranded nucleic acid target under the combined action of the nicking incision enzyme and polymerase and carrying out efficient specificity amplification on the target nucleic acid employing two amplification products and a replacement primer in an isothermal condition. Through selection of one amplification primer and an amplification template annealing region, efficient specificity amplification of the target nucleic acid can be finished by only three primers; the 3' end of the template is completely complementary with the primer; the specificity is improved; meanwhile, the problem of refractory amplification due to the fact that the replacement primer occupies the complementary position of the amplification primer in the prior art is avoided; the amplification primer can also be designed into a form of a molecular beacon; and only correctly amplified target sequence can be annealed together with the primer in the form, and generates subsequent fluorescence signals, so that the method has relatively good specificity.

Disclosed is a novel isothermal nucleic acid amplification method enabling inexpensive and simple and easy detection. The method includes introducing nicking enzyme recognition sequences into an analysis target nucleic acid using nicking enzyme recognition sequence-containing primers and isothermally amplifying a specific region of the target nucleic acid using the primers, a nicking enzyme and a DNA polymerase having strand displacement activity.

The invention belongs to the field of DNA (deoxyribonucleic acid) biosynthesis, and in particular relates to a method for synthesizing a short single strand deoxyribonucleotide probe and application thereof. The short single strand deoxyribonucleotide probe can be used for detecting small non-coding ribonucleic acid (RNA) such as micro ribonucleic acid. The method comprises the following steps of: (1) preparing plasmids recombined with template DNA; (2) cutting and connecting the template DNA, performing enzyme digestion on the recombined plasmids to obtain the template DNA, and connecting the template DNA end to end to form a ring; (3) cutting a single strand, performing rolling-circle replication, cutting one strand of the template DNA by using nickase, adding polymerase to perform rolling-circle replication by taking annular DNA with a nick as a template, and amplifying to obtain a DNA single strand containing a neck ring-probe structure; and (4) preparing target short single strand DNA, cutting the product of the step (3) by using a type II incision enzyme to form a target single strand DNA probe and a byproduct DNA, and separating and purifying through PAGE (polyacylamide gel electrophoresis) to obtain a single strand DNA probe without mutation. The short single strand deoxyribonucleotide probe has wide applications in the fields of detection of small non-coding RNA and diagnosis and treatment of small non-coding RNA related diseases.

The invention discloses a dual-ring hairpin probe mediate label-free strand displacement amplification method for detecting bleomycin. The method includes the steps that when bleomycin exists, a dual-ring hairpin probe fractures at a recognition site, and a triggering sequence is released; the triggering sequence and a ring part of a signal probe are combined and subjected to a strand displacement amplification reaction under the effect of a polymerase and a nicking enzyme, and finally a great number of G-four-chain sequences are generated. At the same time, a primer chain extends to open the signal probe, and therefore the G-four-chain sequences packaged in the neck of the signal probe can be exposed. Finally, NMM molecules are bound to the G-four-chain sequences to generate fluorescence signals, and bleomycin is quantified through the detected fluorescence signals. As the triggering sequence is designed at the neck of the dual-ring hairpin probe, background signals of a detection system are reduced; by combining bleomycin cutting and the strand displacement amplification reaction, bleomycin can be detected sensitively, and the detection limit is 0.34 nm. The method has the advantages of being easy to operate, free of labeling and good in specificity.

The invention provides a method for realizing genome editing and accurate and targeted knock-in of genes in fishes. A technology of producing DNA single-chain gap based on nicking enzyme is utilized,assisted with a set of homologous recombination repair factor RecOFAR, so as to realize high-efficient, accurate and targeted integration of genome editing and gene knock-in in the fishes. The problems that a current existing method is low in efficiency, and random mutation efficiency of target points is higher are overcome.

This disclosure provides methods and compositions for targeted mutagenesis of specific genes in a bacterial strain. By inducibly over-expressing error-prone polymerases such as Pol IV or Pol V in conjunction with nickase in a bacterial strain, and housing the targeted gene(s) on an episome or plasmid which contains one or more nickase recognition sequences, the targeted gene(s) can be selectively mutated at rates significantly greater than genes contained on the chromosome. The methods disclosed herein are useful for engineering desirable bacterial phenotypes and novel strains, including for example strains useful for treating or degrading waste and/or environmental contaminants, for optimizing bioprocesses, and for converting low-value feed-stock into value-added products.

The invention discloses an isothermal amplification nucleic acid detection method based on helicase and nicking enzyme. The isothermal amplification nucleic acid detection method comprises the following steps that S1, a specific amplification primer and a Taqman probe with nicking enzyme recognition sites are designed according to a target nucleotide sequence; S2, a sample nucleic acid is subjected to enzyme treatment to obtain a single-chain nucleic acid template; S3, the Taqman probe obtained in the step S1 and the single-chain nucleic acid template obtained in the step S2 are specifically bonded to obtain a specific bonding product, the specific bonding product is digested by using the nicking enzyme, and real-time detection of fluorescence signals is achieved. The helicase and nickingenzyme are used jointly, the Taqman probe with nicking enzyme recognition sites is designed, a large number of nucleic acid products do not need to be amplified, only an appropriate amount of single-chain templates is needed, and then detection can be performed by using the nicking enzyme to constantly cut the probe bound with recognition sites, the shortcoming of lab pollution likely caused by isothermal amplification can be avoided, and the isothermal amplification nucleic acid detection method is simple and quick to operate and suitable for on-site quick detection.

The present invention relates to the constitution method of one kind of linear expression vector, and especially relates to the cloning-free constitution method of one kind of linear expression vector. The method includes introducing nickase identifying gene in the primer design, and nickase enzyme incision and nucleic acid exoenzyme degradation after DNA segment proliferation to form connected segment with protruding end and connecting the segments to obtain linear expression vector. The method of the present invention has greatly lowered constitution cost of linear expression vector and the wide application of the constitution method in scientific research and production can provide technological platform for gene expression and protein function research.

The invention provides a method for isothermal amplification of a nucleic acid target sequence. The method is suitable for the combined reaction of double-stranded DNA, single-stranded DNA and single-stranded RNA, including nickase and strand displacement enzyme, three primers and one probe are adopted during the detection of the double-stranded DNA and the single-stranded DNA, and three primers and one probe or two primers and one probe can be adopted during the detection of the single-stranded RNA. The probe is a molecular beacon, does not degrade in the amplification process, is only used for specifically binding with a target fragment, provides a fluorescence signal and guarantees the specificity of the reaction. According to the invention, a result is judged in real time by adopting the beacon probe which is not overlapped with the primer in the binding area on the target sequence, and beacon probe binding target sequence has very high specificity; meanwhile, a tube is not opened after the reaction, so that false positive is further avoided; the reaction is carried out at a constant temperature, the consumed time is short, the detection can be completed within 8 minutes, and the POCT detection requirements are met better.

The invention discloses a method for improving plant gene replacing efficiency. The method comprises the following steps of guiding esgRNA, a Cas9 nicking enzyme, a screening agent resistant protein and donor DNA into a purpose plant, enabling the esgRNA to target a DNA fragment A target point sequence, performing coexpression on the Cas9 nicking enzyme and the screening agent resistant protein, wherein the donor DNA sequentially comprises the DNA fragment A target point sequence, a DNA fragment B and a DNA fragment A target point sequence, the DNA fragment B is DNA molecules obtained throughperforming mutation of one or several basic groups on the DNA fragment A, under the guidance of the esgRNA, the Cas9 nicking enzyme produces single-stranded DNA nicking at the DNA fragment A target point sequence in the purpose plant genome and the DNA fragment A target point sequence in the donor DNA, through an in vivo repair mechanism of the purpose plant, the DNA fragment A in the purpose plant genome is replaced with the DNA fragment B, so that plant gene replacement is realized.

The invention provides a method for generating a single-stranded product in an isothermal amplification system. According to the method for generating the single-stranded product, the isothermal amplification system comprises two pair of primers including F1, R1, BF and BR, wherein each of F1 and B1 includes three parts: a fixed region close to a 5' terminal of the primer, a recognition region which is close to a 3' terminal and capable of specifically bound with a template, and a nickase region between the fixed region and the recognition region. The method has the beneficial effects that the reaction speed is high, the cost is low, the operation is simple and convenient, a single-stranded DNA amplified product can be generated by virtue of an isothermal amplification method, and the technical defects in the background art are overcome.

Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b) using a nicking enzyme to cause a nick at each of said nicking sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands to as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to shuttling, a plurality of times, between an upper temperature and a lower temperature during the amplification process of steps (b)-(d), wherein at the upper temperature, one of said polymerase or nicking enzyme is more active than the other of said enzymes, such that there is a disparity in the activity of the enzymes, and at the lower temperature the disparity in the activity of the enzymes is reduced or reversed.